Antimicrobial peptides are a promising class of new antibacterial agents, particularly due to their potent activity against bacteria resistant to conventional antibiotics. Only a few such peptides, e.g., polymyxins, have been in clinical practice, and to date polymyxins are the only antibiotic to which multidrug-resistant Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii remain susceptible. Antimicrobial peptides that structurally differ from the cyclic lipopeptide polymyxins are now at various stages of clinical trials as systemic and topical agents, e.g., cationic 1-helical pexiganan and 2-sheet plectasin. However, recent studies have demonstrated that the use of antimicrobial peptides in clinics will eventually induce a high level of bacterial resistance to them. In its worst scenario, bacteria normally controlled by human endogenous antimicrobial peptides may cause unmanageable infections. Our goal in this project is to identify and characterize genetic determinants of bacterial intrinsic resistance to human antimicrobial peptides such as cationic 2-sheet neutrophil peptide-1 (HNP-1) and anionic dermcidin-1, as a prerequisite to assessing the potential risk of bacterial resistance to them and overcoming such resistance. In Gram-negative pathogens, mechanisms of resistance to these classes of antimicrobial peptides are virtually unknown. To expedite the discovery of resistance determinants, we have developed a new microarray-based method, monitoring of gene knockouts (MGK). MGK allows simultaneous analysis of the relative abundance of thousands of mutants grown in a culture. Our preliminary studies demonstrate that this method works very well and a pilot experiment using MGK has identified genes previously unknown to be involved in HNP-1 resistance. Based on this powerful method, we propose the following specific aims: (1) to identify genetic determinants of cationic 2-sheet HNP-1 resistance;(2) to identify genetic determinants of anionic dermcidin resistance;and (3) to characterize genes involved in HNP- 1 and dermcidin resistance. Successful accomplishing of this project will set the stage for in-depth understanding of mechanisms of bacterial resistance to antimicrobial peptides as planned in a follow-up RO1 project. Moreover, as bacterial pathogens defective in antimicrobial peptide resistance are attenuated in animal infections, a comprehensive list of resistance determinants may help understand their contribution to bacterial pathogenesis and may provide insights into new anti-infective strategies. Antimicrobial peptides are a promising class of new antibacterial therapies due to their potent bactericidal activity against bacteria resistant to conventional antibiotics, and they are now at various stages of clinical trials for systemic and topical use. Thus, the understanding of bacterial natural resistance to these peptides is important as a prerequisite to assessing potential emergence of bacteria highly resistant to them in clinical settings and possibly developing measures to overcome such resistance.